The proposed project has two basic objectives. First, we wish to characterize the physiological binding of several organic acids to renal cortical homogenate. Second, we wish to further characterize organic acid binding in subcellular fractions. Several different membrane fractions exist in the microsomal fractions. These include the plasma membranes, and a characterization of their binding properties will further clarify the mechanism of action and localization of the carrier-mediated transport of organic acids. We plan to use millipore filtration to separate protein from fluid. Millipore filtration offers the advantage of a time course, therefore kinetic, and a temperature dependency study not feasible with other methods. Homogenates obtained from slices of rabbit renal cortex will be placed in Cross-Taggart media. We intend to examine the physiological binding characteristics of organic acids to renal cortical homogenates as a function of time, ionic environment, temperature, pH and, investigate the action of metabolic and competitive inhibitors on this binding. Bromcresol green (BCG), phenol red (PSP) and para-aminohippurate (PAH) were chosen because they utilize a common renal transport system and, because they have a broad array of inhibitory constants. We then plan to study the binding properties of PAH, PSP and BCG to various subcellular fractions of renal cortex. These fractions will be obtained by differential ultracentrifugation and their purity will be ascertained by use of electron microscope examination and by estimation of enzymes characteristic for each fraction. Binding characteristics of BCG, PSP and PAH to these purified fractions will clarify the site and mechanism by which they are transported.